Western Blot Analysis of BM-MSCs

BMSCs were analysed by western blotting based on our previous study [13 (link)]. For the preparation of total cell lysates, BMSCs were lysed in RIPA buffer (P0013B; Beyotime, Shanghai, China) at 4 °C. The samples were centrifuged, and the protein concentrations were checked using the Enhanced BCA Protein Assay kit (P0010S, Beyotime). The supernatants were separated on a 12% SDS-PAGE gel and subsequently transferred to a PVDF membrane (Immobilon-P Membrane, Millipore, USA). The membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and then incubated with the appropriate primary antibodies overnight at 4 °C. Then, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at 24 °C. Immunoreactive bands were revealed by the BeyoECL Plus reagent (P0018, Beyotime) using the Photo-Image System (Molecular Dynamics, Sunnyvale, CA, USA). β-Actin was used as a loading control for the western blotting analysis. The following antibodies were used to analyse protein expression levels by western blotting: HJURP (ab224076, Abcam), GDF15 (ab39999, Abcam), CDKN1A (ab109520, Abcam) and p53 (ab1101, Abcam).